BubR1 autophosphorylation is necessary for metaphase chromosome alignment. (A) Schematic representation of BubR1 siRNA, rescue transfection, and chemical inhibitor treatment protocol. IF, immunofluorescence. (B) Replacing endogenous BubR1 with a nonphosphorylatable mutant (T608A) or a kinase-inactive (KD) mutant results in severe chromosome misalignment. Cells were imaged for mCherry-BubR1 (mCherry), ACA, microtubules (MT; by an antitubulin antibody), and DNA (visualized by DAPI). Bar, 10 µm. (C) The percentage of mitotic cells with fully aligned chromosomes was analyzed in cells transfected with BubR1 siRNA and the siRNA-resistant mCherry-BubR1 constructs as indicated. For each condition, >200 mitotic cells from three repeated experiments were counted to quantify the percentage of cells. Error bars represent standard error; n = 3. (D) Quantitation of the normalized integrated intensity of mCherry-BubR1 signals against ACA signals at unattached and attached kinetochores from ≥10 mitotic cells each from two experimental repeats. Error bars represent standard error. WT, wild type; TA, T608A; TE, T608E.